Venezuelan (VEEV), Western (WEEV), and Eastern Equine Encephalitis viruses (WEEV), are emerging pathogens that cause human encephalitis, yet there are no approved human vaccines or antiviral agents for treating or preventing any alphaviruses infection. Our research objectives include the development of a broad spectrum antiviral clinical candidate against these encephalitic alphavirus infections in humans. The aims of Research Project 1 within the U19 Center of Excellence for Encephalitic Alphavirus Therapeutics (CEEAT) program focus on the lead optimization activities of two distinct small molecule scaffolds with prophylactic and therapeutic in vivo efficacy. Specifically, the project will improve lead prototypes through iterative multi-parameter medicinal chemistry optimization, guided by ADME/PK assessment, cell culture and mechanistic studies (Research Project 3) and in vivo assessments (Research Project 2) for exposure, optimum dosing and safety in higher order species (i.e., rats and non-human primates). The project will manage all synthetic chemistry needs including medicinal chemistry, non-GMP scaling and validation of analogs, point of compound distribution to CEEAT labs, and implementation of process manufacturing improvements prior to CRO engagement for API synthesis to advanced GLP toxicological studies. Additionally, formulation and stability analyses will be conducted, in collaboration with the UW-Madison Zeeh Formulation Station. Several activities pertaining to API and formulation generation and characterization will be accompanied by appropriate certification and analyses in accord with pre-IND requirements, coordinated and overseen by product development and regulatory consultants within the CEEAT structure.